The main gap in our knowledge of evolution is understanding the detailed biochemical processes by which organisms already well adapted to their environment acquire new functions. It is the purpose of this project to fill that gap by providing a solid experimental basis for a general theory of evolution of new functions. My model system will continue to be the artificial evolution of lactose utilization in the bacteria E. coli and Klebsiella. I have already shown that under intensive selection E. coli evolves a new beta-galactosidase (ebg), which is biochemically and genetically distinct from the classical enzyme specified by the lacZ gene. My preliminary evidence suggests that Klebsiella evolves a completely new lactose utilization system, but that it does so in a way very different from E. coli. By monitoring the changes which occur in the evolution of lactose utilization in these two systems I shall eventually be able to describe the kinds of mutations which lead from the progenitor genes to the genes permitting growth on lactose. Ultimately I hope to be able to describe the variety of pathways available for the evolution of this new function, and to understand the "rules" for employing particular evolutionary pathways. In the course of this study I shall: (1) continue studies of the ebg system per se; (2) study the homology between ebg and the classical lac operon; (3) study the experimental evolution of lactose utilization in Klebsiella; including (a) studies of a phospho-beta-galactosidase (previously unknown in gram negative bacteria), (b) a novel lactose transport system; and (c) the fate of lactose metabolites in this unique system. I feel that the project will contribute significantly to understanding the processes of evolution; and to developing the techniques and concepts for artificially evolving new enzymes of medical, ecological, and industrial importance.